Project Summary/Abstract Immunotherapy is proving to be a turning point in the treatment of cancer. Notwithstanding these encouraging signs, predictors of a patient's response to immunotherapy remain to be defined, as actual responses are heterogeneous and difficult to study in real-time. The question is: what is behind the success or failure of immunotherapy? To tackle this issue, we need new methods to explore the microenvironment of tumors non- invasively and to enumerate the relevant cell types. Imaging the extent of infiltration of immune cells into tumors and identifying the relevant lymphocyte subsets may therefore be an attractive means of stratifying responders and non-responders. Non-invasive monitoring via immuno-PET imaging, as proposed in this application, could therefore change how therapies are applied and assessed, to the benefit of many patients. Specifically, the study proposed here will focus on developing modified single domain antibodies to image different subsets of immune cells, and peptide-MHC complexes to image antigen-specific T cells. The proposed imaging tools will allow us to noninvasively acquire a more comprehensive understanding of the immune infiltrate status in the tumor microenvironment in response to therapy. The results may help us to better understand how the tumor microenvironment is shaped to be either more immunosuppressive or anti-tumor. This, in turn, may help improve or customize treatments. Ultimately, we may find predictive criteria. Because the basic methodology proposed here is easily translated to a human setting, it is envisioned that this approach will ultimately find clinical application. Dr. Mohammad Rashidian (PI of the grant) is a postdoctoral fellow in the laboratory of Dr. Hidde Ploegh. He received his Ph.D. from the University of Minnesota in the chemical biology program where he worked on protein and antibody labeling approaches. He published five first author papers during Ph.D., and developed several methods for rapid and efficient site-specific protein and antibody modifications. In the Ploegh lab, Dr. Rashidian had pioneered an approach that allows whole-body imaging and tracking anti-tumor immune responses in real- time. Building off of this finding, he will now examine the extent to which it is possible to image anti-tumor immune responses. His Ph.D. training has prepared him for the necessary chemical approaches, and in the Ploegh lab Dr. Rashidian has rapidly familiarized himself with the immunology and cancer immunotherapy, as applied to VHHs and their use in imaging of immune responses. During the remaining of his time in Dr. Ploegh's lab and until he secures an independent faculty position, he will focus on developing the new PET agents and also focus on enhancing his expertise in immunology. The Karp/BCH and the Ploegh lab are very well equipped to support his project and to facilitate his cancer immunology training. In the long term, Dr. Rashidian will focus on the root cause of heterogeneous response of immunotherapy. He plans to launch an independent research career at an academic institution where he can continue and focus on his research goals and train graduate students and post-docs in his lab.!